RNA and DNA elicit inflammatory responses which are critical for anti-microbial immunity. DNA is a potent trigger of inflammatory cytokine and type I IFN gene transcription, as well as caspase-1-mediated processing of the pro-inflammatory cytokines IL-1 and IL-18. Growing evidence indicates that DNA recognition is central not only to anti-microbial host defenses but is also a major contributor to the adjuvant activity of DNA vaccines as well as the pathology associated with autoimmune diseases such as Systemic Lupus Erythematosis. It is therefore critical that we understand the molecular basis of DNA recognition. Recent work from our group has implicated the PYHIN family members Absent in melanoma-2 (AIM2) and the interferon-inducible protein (IFI16) as sensors of microbial DNA. Both proteins bind DNA via HIN domains. AIM2 engages ASC via a pyrin domain to form a caspase-1 activating inflammasome while IFI16 activates a cytosolic signaling pathway involving Stimulator of IFN genes (STING), TANK binding kinase-1 (TBK-1) and interferon regulatory factor 3 (IRF3) to regulate transcription of type I IFN genes. Knockdown of IFI16, or its murine ortholog p204 prevents viral DNA from triggering IRF3 activation and IFNB gene induction, while not affecting responses to cytosolic RNA. Recent evidence also indicates that IFI16 can form an inflammasome in responses to nuclear sensing of Kaposi's Sarcoma Herpes Virus infection. Additional work from our laboratories has also implicated the DEAD box helicase DEAD box protein 3x (DDX3x) in the TBK1/IRF3 signaling pathway important for both RNA and cytoplasmic DNA pathways (including for IFI16), but its role in innate immunity remains to be fully characterized. The underlying hypothesis to be tested in this proposal is that IFI16 plays a central role in innate immunity and host-defense to microbial pathogens by regulating inflammatory responses and that DDX3x is an important downstream component of the IFI16 signaling pathway. We propose to explore the molecular mechanisms of IFI16 activation and define the role of IFI16 and DDX3x in anti-viral host-defenses. This project will increase our understanding of how the innate immune system senses pathogens, leading to novel therapeutic targets.